Distinguishing True HPV-Driven Head and Neck Squamous Cell Carcinoma by QuantiGene™ Molecular-Profiling-Histology Assay from HPV-Unrelated Cases Allows for More Precise Prediction of Prognosis and Possibly Has Therapeutic Implications

Background: Head and neck squamous cell carcinoma (HNSCC) with discordant diagnostic pat-terns of HPV⁺/p16⁻ or HPV⁻/p16⁺ correlate with worse prognosis. This study aims to identify truly HPV-driven HNSCCs using mRNA profiling by QuantiGene Molecu-lar-Profiling-Histology (QG-MPH) assay for more precise etiological diagnosis. Methods Of 97 FFPE samples analyzed, 68 were valid for HPV DNA detection by PCR and quantification of HPV E7 and p16ᴵᴺᴷ⁴ᵃ mRNA using QG-MPH. p16ᴵᴺᴷ⁴ᵃ mRNA expression was then compared with p16 protein expression by immunohistochemistry (p16 IHC). Results Among the 68 cases, 26 (38.2%) showed increased high-risk HPV E7 mRNA expression (hrHPV E7 mRNA⁺), while 37 (54.4%) were hrHPV DNA⁺. The concordance between HPV DNA and mRNA status was 70.1%. Notably, 79.2% of E7 mRNA⁺ cases were p16 IHC⁺, compared to 55.9 % of DNA⁺ cases, demon-strating better concordance between HPV E7 mRNA⁺ status and p16 mRNA expression plus p16 IHC positivity. All patients (19/19) in the HPV E7 mRNA⁺/p16 IHC⁺ group survived the 5-year follow-up, compared to 59.5% (22/37) in the HPV E7 mRNA⁻/p16 IHC⁻ group (p = 0.001). Specifi-cally, the OS rate was 57.1 % (8/14) in the group with discordant HPV DNA and p16 IHC results, compared to 40% (3/5) in the group with discordant HPV E7 mRNA and p16 IHC results. These findings highlight the better outcome for the transcriptionally active HPV cases and indicate the prognostic disadvantage for patients with discordant categories and the advantages for incorpo-rating the molecular mRNA profiling by QG-MPH to p16 IHC in making therapeutic decision and predicting patient outcomes in HNSCC. Conclusion QG-MPH quantification of E7 and p16ᴵᴺᴷ⁴ᵃ mRNA is more precise in identifying truly HPV-driven HNSCC than HPV DNA testing alone or with p16 IHC, enabling improved prognosis prediction and therapeutic deci-sion-making.